Annette M. Griffin

Computer Analysis of Sequence Data

GCG: Fragment Assembly Programs. GCG: Drawing Linear Restriction Maps. GCG: Drawing Circular Restriction Maps. GCG: Displaying Restriction Sites and Possible Translations in a DNA Sequence. GCG: Assembly of Sequences into New Sequence Constructs. GCG: Comparison of Sequences. GCG: Production of Multiple-Sequence Alignment. GCG: Database Searching. GCG: Pattern Recognition. GCG: Translation of DNA Sequence. GCG: Analysis of Protein Sequences. GCG: The Analysis of RNA Secondary Structure. GCG: Preparing Sequence Data for Publication. MicroGenie: Introduction and Restriction Enzyme Analysis. MicroGenie: Shotgun DNA Sequencing. MicroGenie: Translation. MicroGenie: Protein Analysis. MicroGenie: Homology Searches. PC/GENE: Sequence Entry and Assembly. PC/GENE: Restriction Enzyme Analysis. PC/GENE: Translation and Searches for Protein Coding Regions. PC/GENE: Sequence Comparisons and Homologies. PC/GENE: Database Searches. PC/GENE: Searches for Functional Sites in Nucleic Acids and Proteins. Using the FASTA Program to Search Protein and DNA Sequence Databases. Converting Between Sequence Formats. Obtaining Software via INTERNET. Submission of Nucleotide Sequence Data to EMBL/GenBank/DDBJ. Index.

The cpsABCDE genes involved in polysaccharide production in Streptococcus salivarius ssp. thermophilus strain NCBF 2393.

A 4074-bp EcoRI fragment of Streptococcus salivarius ssp. thermophilus (S. thermophilus) chromosomal DNA containing genes involved in exocellular polysaccharide (EPS) was identified and cloned. The nucleotide sequence of this fragment was determined and found to contain one partial and four complete open reading frames. These were designated cpsA, cpsB, cpsC, cpsD and cpsE and encoded proteins of > 130, 243, 230, 246 and 455 amino acids, respectively, that showed homology with the genes of the cps cluster, involved in polysaccharide biosynthesis, in Streptococcus pneumoniae Type 19F. The cpsA gene is predicted to encode a transcriptional regulator, while cpsC and cpsD are predicted to encode proteins involved in polysaccharide polymerization and export. The cpsE gene is likely to encode the phosphate-prenyl glycosyl-1-phosphate transferase catalyzing the first step in polysaccharide biosynthesis in S. thermophilus. Southern blot analysis revealed that cpsE is found only in polysaccharide producing strains of S. thermophilus.

DNA sequencing protocols.

DNA Sequencing. M13 Cloning Vehicles: Their Contribution to DNA Sequencing. Cloning into M13. Transfection of E. coli with M13 DNA. M13 Phage Growth and Single Strand DNA Preparation. M13 Phage Growth and DNA Purification Using 96 Well Microtiter Trays. Generation of Random Fragments by Sonication. Generation of a Nested Set of Deletions Using Exonuclease III. Sequential Deletions of Single-Stranded DNA: Cyclone Sequencing. Subcloning for DNA Sequencing. Sequencing Using Custom Designed Oligonucleotides. Dideoxy Sequencing Reactions Using Klenow Fragment DNA Polymerase 1. Dideoxy Sequencing Reactions Using T7 Polymerase. Dideoxy Sequencing Reactions Using Sequenase Version 2.0. Dideoxy Sequencing Reactions Using Taq Polymerase. Pouring Linear and Buffer-Gradient Sequencing Gels. Electrophoresis of Sequence Reaction Samples. Plasmid Sequencing. Direct Sequencing of Inserts Cloned into Lambda Vectors. Cosmid Sequencing. Genomic Sequencing. Sequencing of Double-Stranded PCR Products. Sequencing Double-Stranded Linear DNA with Sequenase and [a-35S]-dATP. Solid Phase PCR Sequencing of Biotinylated Products. Cycle Sequencing. Direct Blotting Electrophoresis. Multiplex DNA Sequencing. DNA Sequencing by Chemiluminescent Detection. Reverse Sequencing of M13 Cloned DNA. Terminal Labeling of DNA for Maxam and Gilbert Sequencing. DNA Sequencing by the Chemical Method. DNA Sequencing by Chemical Degradation Using One Two and Four Different Fluorophores. Linear Amplification Sequencing with Dye Terminators. Sequencing Reactions for the Applied Biosystems 373A Automated Sequencer. Sequencing Reactions for the ALF (EMBL) Automated Sequencer. Sequencing Using the DuPont Genesis% 2000 DNA Analysis System. The Use of Robotic Workstations in DNA Sequencing. Index.

Structure and conformation of a novel genetically engineered polysaccharide P2

A new exocellular polysaccharide (P2) has been produced by the manipulation of a glycosyl transferase gene (aceP) involved in the biosynthesis of the polysaccharide acetan by the bacterium Acetobacter xylinum strain CKE5. The P2 polysaccharide has been studied by methylation analysis, reductive cleavage, and 1H and 13C NMR spectroscopy. The data are consistent with the structure predicted when the aceP gene is deactivated: [Molecular structure: see text]. The effect of cooling on proton NMR line width indicates a coil-helix transition in P2 at about 70 degrees C.

Generation of a novel polysaccharide by inactivation of the aceP gene from the acetan biosynthetic pathway in Acetobacter xylinum

The acetan biosynthetic pathway in Acetobacter xylinum is an ideal model system for engineering novel bacterial polysaccharides. To genetically manipulate this pathway, an Acetobacter strain (CKE5), more susceptible to gene-transfer methodologies, was developed. A new gene, aceP, involved in acetan biosynthesis was identified, sequenced and shown to have homology at the amino acid level with beta-D-glucosyl transferases from a number of different organisms. Disruption of aceP in strain CKE5 confirmed the function assigned above and was used to engineer a novel polysaccharide with a pentasaccharide repeat unit.

Cloning and DNA sequence analysis of the serC-aroA operon from Salmonella gallinarum ; evolutionary relationships between the prokaryotic and eukaryotic aroA-encoded enzymes

The serC-aroA operon of Salmonella gallinarum was isolated from a gene library using a labelled oligonucleotide probe and by complementation of an aroA Escherichia coli strain. The nucleotide sequence of a 2.6 kbp fragment was determined. The predicted amino acid sequence of the aroA gene product was compared to the equivalent sequence from ten other organisms. Computer-generated evolutionary trees clearly divide the eleven sequences into four different groups: Gram-negative bacteria, Gram-positive bacteria, fungi and plants. These trees depict a close evolutionary relationship between the sequences from Gram-negative bacteria and higher plants.

Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum: nucleotide sequence analysis of the aceB aceC aceD and aceE genes

Sequence analysis of a 5.323 kb chromosomal DNA fragment from Acetobacter xylinum involved in the biosynthesis of the exopolysaccharide acetan, revealed the presence of four ace genes designated aceB, aceC, aceD and aceE. Comparison of translated gene sequences to the databanks was used to assign putative gene functions. AceB displayed strong homology to a glucose-diphosphoprenyl beta, D-glucose transferase from Xanthomonas campestris, while AceC was homologous to a cellobiosyl-diphosphoprenyl alpha, D-mannose transferase from the same organism. Thus these genes encode enzymes catalyzing the second and third steps of the acetan biosynthetic pathway. AceD and AceE were homologous to ExoP and ExoT respectively from Rhizobium meliloti and are likely to be involved in acetan polymerization and export.

Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum

We have identified, cloned and sequenced an 8422 base pair fragment of Acetobacter xylinum genomic DNA containing part of the acetan biosynthetic gene cluster. Computer analysis of the nucleotide sequence data generated revealed the presence of six open reading frames. Comparison of the translated sequences of putative genes to the amino acid sequences of genes from other organisms was used to assign functions to the aceA, aceC and manB genes. These genes were predicted to encode a UDP-glycosyl transferase, a GDP-mannosyl transferase and a phosphomannose isomerase/GDP-mannose pyrophosphorylase, respectively.